Roles of Homologous Recombination in Processing DNA Lesions
8 9 Scope of the Thesis Scope of the Thesis The abundance of DNA damaging agents poses a constant threat for genome stability. Therefore, cells have evolved multiple mechanisms to repair their DNA. The variety in possible DNA lesions that can occur require specified repair mechanism with the ability to remove particular types of lesions. For example, nucleotide excision repair removes lesions generated by UV-light whereas homologous recombination repairs DNA double-stranded breaks induced by ionising radiation. However, the same type of DNA lesion can be present in the various DNA contexts, resulting in difficulties to detect the damage by proteins of a specified repair system. For example, the UV-light-induced lesion generated at the site of replication fork might not be recognised by nucleotide excision repair proteins because of the DNA structure in which the damage is present, i.e. in double-stranded versus single-stranded DNA. Thus in this case, modification of DNA organisation around the lesion, required for damage recognition, can be performed by proteins with the ability to change DNA conformation. Some of the proteins of homologous recombination, such as Rad54, have the ability to alter DNA topology, therefore they could act upon DNA lesions induced by UV-light at sites of replication forks. Resulting modification of DNA arrangement neighbouring the lesion allows damage detection and subsequent removal by nucleotide excision repair proteins. This thesis describes the cellular behaviour of the mammalian DNA double-stranded break repair protein, Rad54 upon induction of DNA lesions by UV-light. Additionally, the effect on cellular metabolism of inactivation of the Rad54 protein in nucleotide excision repair-deficient cells is discussed. In addition to this study, biochemical and genetic analysis of recently identified Rad54 paralogue, Rad54B, is included in the thesis. Finally, the consequences of incorrect DNA damage repair, reflected by severe clinical cases, are discussed. Chapter 1 provides an overview of mechanisms of replication and homologous recombination as well as presents the established evidence for the possible roles of homologous recombination in supporting replication in both, prokaryotic and eukaryotic cells. Chapter 2 describes the analysis of the functionality of a Rad54 knockin system generated in both nucleotide excision repair -proficient and -deficient mouse embryonic stem cells. Chapter 3 examines the difference in nuclear distribution of the Rad54 protein induced by either ionising radiation or UV-light. Furthermore, the discrepancy in Rad54 foci formation between nucleotide excision repair-proficient and -deficient effect cells in response to UV-light is presented. Finally, UV-light-induced alterations of cell cycle progression of both cell lines are discussed. Chapter 4 demonstrates the effect of inactivation of the Rad54 protein in nucleotide excision repair-deficient cells on the survival, accumulation of structural chromosomal aberrations and the replication restart upon UV-light treatment of these cells. Chapter 5 presents an analysis of the biochemical properties of the mammalian Rad54 paralogue, Rad54B. Additionally, the examination of the sensitivity to DNA-damaging agents of cells and mice deficient in these genes is included. Chapter 6 provides an overview of the possible consequences of malfunction of DNA repair pathways, which may result in chromosomal rearrangements including translocations. Moreover, diagnostic and prognostic applications of specific translocations are considered.
|Keywords||DNA, DNA recombination, nucleotides, proteins|
|Promotor||J.H.J. Hoeijmakers (Jan) , R. Kanaar (Roland)|
|Publisher||Erasmus University Rotterdam|
|Sponsor||Dutch Cancer Society (KWF), Netherlands Organization for Scientific Research (NWO), EU|
Tafel, A.. (2007, May 2). Roles of Homologous Recombination in Processing DNA Lesions. Erasmus University Rotterdam. Retrieved from http://hdl.handle.net/1765/18603